Structural studies of HCN channels in health and disease

项目摘要 此项目的目标是了解位于以下位置的域如何管理HCN通道的门控 蛋白质的胞质、胞内部分。HCN通道调节心脏的起搏，并且 大脑中的神经元兴奋性。它们具有电压门控K+通道的一般结构，但 在膜超极化时被激活，并传导向内的去极化电流。重要的是，他们的 门控受cAMP与细胞内环核苷酸结合域(CNBD)直接结合的调节 位于蛋白质的C-末端部分。最近的结构性数据也揭示了 第二，紧接在前面的蛋白质细胞内N-末端部分的高结构结构域 第一个跨膜区(HCN区)。在这项研究中，我们将结合使用结构和 确定HCN结构域在通道选通的调制中的潜在作用的功能方法， 以及C-末端CNBD和N-末端HCN结构域之间相互作用的性质。作为不同的 HCN亚型(HCN1-4)表现出明显不同的生物物理特性，我们将利用这些差异来 确定可能有助于调节HCN通道门控的关键接触者和残基。在目标1中，我们 将使用冷冻电子显微镜(CryoEM)来确定HCN4异构体的结构，在 和不存在cAMP，并将其特征与现有的HCN1低温EM结构进行比较。在目标2中，我们将 在干扰cAMP介导的易化cAMP的配体存在下解析HCN4的结构 通道开放(辅助亚单位TRIP8b，联苯基脲化合物BPU)，并使用结构数据 通过目标1和目标2获得，以便为旨在测试和解释任何推断的功能实验提供信息 结构/功能关系模型。最后，在目标3中，我们将使用类似的组合 研究遗传性癫痫患者中发现的特定HCN1和HCN4突变的功能研究 心律失常。

Project Summary The goal of this project is to understand how the gating of HCN channels is regulated by domains located in the cytoplasmic, intracellular portion of the protein. HCN channels regulate pacemaking in the heart, and neuronal excitability in the brain. They have the general structure of voltage-gated K+ channels, but are activated upon membrane hyperpolarization and conduct an inward, depolarizing current. Importantly, their gating is modulated by the direct binding of cAMP to an intracellular cyclic-nucleotide binding domain (CNBD) located in the C-terminal portion of the protein. Recent structural data have also revealed the presence of a second, highly structured domain in the intracellular N-terminal portion of the protein, immediately preceding the first transmembrane domain (HCN domain). In this study, we will use a combination of structural and functional approaches to determine the potential role of the HCN domain in the modulation of channel gating, and the nature of the interactions between the C-terminal CNBD and N-terminal HCN domain. As different HCN isoforms (HCN1-4) exhibit markedly distinct biophysical properties, we will exploit these differences to identify key contacts and residues that may contribute to modulate the gating of HCN channels. In Aim 1, we will use cryo-electron microscopy (cryoEM) to determine the structure of the HCN4 isoform, in the presence and absence of cAMP, and compare its features to the available cryoEM structure of HCN1. In Aim 2, we will resolve the structure of HCN4 in the presence of ligands that interfere with the cAMP-mediated facilitation of channel opening (auxiliary subunit TRIP8b, biphenylurea compound BPU), and use the structural data acquired through Aims 1 and 2 to inform functional experiments designed to test and interpret any inferred model of structure/function relation. Finally, in Aim 3, we will use a similar combination of cryoEM and functional studies to study select HCN1 and HCN4 mutations found in patients with genetic epilepsy and cardiac arrhythmias.